NRMT: An a-N-methyltransferase

Petkowski, Janusz Jurand, Department of Biophysics, University of Virginia
Minor, Wladek, Department of Molecular Phys and Biological Physics, University of Virginia
Brautigan, David, Department of Microbiology, University of Virginia
Nakamoto, Robert, Department of Molecular Phys and Biological Physics, University of Virginia
Pemberton, Lucy, Department of Microbiology, University of Virginia

The post-translational methylation of alpha-amino groups was first discovered over 30 years ago on bacterial ribosomal proteins L16 and L33, but prior to the work presented here almost nothing was known about the function or enzymology of this modification. Several other bacterial and eukaryotic proteins have since been shown to be alpha-Nmethylated. The Ran guanine nucleotide-exchange factor, RCC1, is the only protein for which any biological function of alpha-N-methylation has been identified. Methylationdefective mutants of RCC1 are known to have a reduced affinity for DNA and cause mitotic defects, but further characterization of this modification has been hindered by ignorance of the responsible methyltransferase. In this work, we show that all fungal and animal N-terminally methylated proteins share a unique N-terminal motif, M-X-P-K, where the initiating Met is cleaved, and the exposed alpha-amino group is mono-, dior trimethylated, and where the X is A, P, or S. Alpha-N-methyltransferase, named N- terminal RCC1 methyltransferase I (NRMTI) can also methylate artificial substrates in vitro in which X is G, F, Y, C, M, K, R, N, Q, or H. Methylation efficiencies of N- terminal amino acids are variable with respect to the identity of X. Here I describe studies which have led to the discovery of the first alpha-N-methyltransferase, (NRMTI). Substrate docking and mutational analyses of RCC1 helped us define the NRMTI recognition sequence and enabled us to identify numerous new methylation targets, including SET (also known as TAF-I or PHAPII) and the retinoblastoma protein (RB). Further in vitro peptide methylation assays and substrate immunoprecipitations showed that the canonical M-X-P-K methylation motif is not the only one recognized by NRMTI. ii We predict that N-terminal methylation is a widespread post-translational modification and that there is interplay between N-terminal acetylation and N-terminal methylation. We also use isothermal calorimetry experiments to demonstrate that NRMTI can efficiently recognize and bind to its fully methylated products. Knockdown of NRMTI recapitulates the multi-spindle phenotype seen with methylation-defective RCC1 mutants, demonstrating the importance of alpha-N-methylation for normal bipolar spindle formation and chromosome segregation.

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PHD (Doctor of Philosophy)
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